Virological characteristics correlating with SARS-CoV-2 spike protein fusogenicity

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike (S) protein is essential in mediating membrane fusion of the virus with the target cells. Several reports demonstrated that SARS-CoV-2 S protein fusogenicity is reportedly closely associated with the intrinsic pathogenicity of the virus determined using hamster models. However, the association between S protein fusogenicity and other virological parameters remains elusive. In this study, we investigated the virological parameters of eleven previous variants of concern (VOCs) and variants of interest (VOIs) correlating with S protein fusogenicity. S protein fusogenicity was found to be strongly correlated with S1/S2 cleavage efficiency and plaque size formed by clinical isolates. However, S protein fusogenicity was less associated with pseudoviral infectivity, pseudovirus entry efficiency, and viral replication kinetics. Taken together, our results suggest that S1/S2 cleavage efficiency and plaque size could be potential indicators to predict the intrinsic pathogenicity of newly emerged SARS-CoV-2 variants.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19).Since the first cases of a novel coronavirus infection were detected in Wuhan, Hubei Province, China, in December 2019 1,2 , the disease spread rapidly over the world and World Health Organization (WHO) declared it a Public Health Emergency of International Concern (PHEIC) as a COVID-19 pandemic on January 30, 2020 3 .Although WHO announced the end of PHEIC on May 5, 2023 4 , the COVID-19 pandemic is not over, thus far causing millions of deaths globally 5 .SARS-CoV-2 displays a long RNA genome of approximately 30 kbp 6,7 , encoding 4, structural proteins [Spike (S), Envelope (E), Nucleocapsid (N), Membrane (M)], 9 accessory proteins (ORF3a, 3b, 6, 7a, 7b, 8, 9b, 9c, and 10), and 16 nonstructural proteins (NSPs; NSP1-16, encoded by the ORF1a and ORF1b genes), respectively [7][8][9] .SARS-CoV-2 S protein fulfills an important role in mediating the virus-target cell membrane fusion, thereby triggering viral entry into the target cells 10,11 .After its translation, cellular proteases (e.g., Furin in the Golgi apparatus of the infected cells) cleave the S protein into two subunits, S1 and S2 10 .This S1/S2 cleavage is important for SARS-CoV2 pathogenicity as SARS-CoV-2 lacking the furin cleavage site in the S protein exhibits attenuated viral pathogenicity in cell line, mouse, and hamster models 12 .The S protein is assembled as a trimer, inserted into viral particles along with the other viral components.The S1 and S2 subunits bind noncovalently and are exposed on the viral surface until meeting the target cells.To enter the target cells, the viral S protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2) have to interact 13 .The receptor-binding domain engagement in the S1 subunit with ACE2 induces conformational changes in the S protein, leading to the S2' site cleavage in the S2 subunit and fusion peptide insertion into the target cell membrane 10 .Next, the 6bundle helix is formed by heptad repeats (HR) 1 and 2 of the S2 subunit as an indispensable fusion step, creating a fusion pore that facilitates genetic material transfer into the host cells.
The S2' site cleavage in the S2 subunit depends on the following SARS-CoV-2 entry routes 10 .
First, the endosome-mediated entry pathway.Upon the S1 subunit-ACE2 interaction, a virus-ACE2 complex is internalized into the target cells by forming an endosome, where cathepsin L cleaves the S2 subunit S2' site 10 .Second, the target cell surface, using transmembrane protease serine 2 (TMPRSS2) for the S2 subunit S2' site cleavage 10,14 .SARS-CoV-2 is continually evolving, with mutations appearing in its RNA genome since its discovery in 2019 6,15 .These S gene mutations influence transmissibility, pathogenicity, and vaccine-and viral infection-induced immune response resistance 11,14,16- 19 .Multiple SARS-CoV-2 variants have emerged during the pandemic, certain among them predominantly spreading across the world.Initially, the Wuhan-Hu1 strain was outcompeted by the B.1 lineage (harboring the D614G mutation in the S protein), a potential ancestor of all recent variants 11,15,17 .Then, WHO defined the Alpha, Beta, Gamma, Delta, and Omicron BA.1, BA.2, and BA.5 variants as variants of concern (VOCs) as well as Lambda and Mu as variants of interest (VOIs) 11,15,17 , being already de-escalated from the VOC and VOI lists.
Efforts to describe the SARS-CoV-2 S protein features revealed that the S protein fusogenic potential of the SARS-CoV-2 variants is closely associated with their pathogenicity, determined using a hamster model without immunity against vaccines and viral infection (hereafter referred to as intrinsic pathogenicity) [20][21][22][23][24] .For example, the Delta variant possesses relatively more fusogenic S protein than the Omicron BA.1 and B.1.1 variants 21,23 .SARS-CoV-2 variants display higher intrinsic pathogenicity associated with the S proteinmediated fusogenicity strength [20][21][22][23][24] .However, the association of S protein-mediated fusogenicity with other virological parameters remains to be fully determined.In this study, we investigate the virological parameters of eleven SARS-CoV-2 variants correlating with S protein fusogenicity.

S protein fusogenicity of eleven SARS-CoV-2 variants
SARS-CoV-2 S protein mediates the virus-infected cell membrane fusion 10,11 .To quantitatively monitor the fusion kinetics between effector cells expressing S protein from eleven SARS-CoV-2 variants (including the previous VOCs and VOIs) and target cell membranes, we performed S protein-mediated membrane fusion assay in Calu-3/DSP1-7 cells 20,[22][23][24][25][26][27] .Although the Alpha, Beta, Gamma, Delta Lambda, Mu, and BA.5 S protein expression levels were lower than that of the B.1.1 S protein (harboring the D614G mutation) on the transfected HEK293 cell surface, the remaining S protein levels were comparable (Fig. 1A).Compared to B.1.1 S protein, the Wuhan S protein exhibited lower fusogenicity, while that related to the Alpha, Beta, Gamma, Delta, Lambda, and Mu S proteins was significantly higher (Fig. 1B).Notably, the Delta S protein exhibited profound fusogenicity 21,23 , being the highest of all tested S proteins (Fig. 1B).The BA.1 and BA.2 S proteins exerted lower fusogenicity compared to the B.1.1 S protein while that of the BA.5 S protein was not significantly different (Fig. 1B).These data indicate that SARS-CoV-2 S protein fusogenicity is different before and after Omicron variant emergence.

Correlation of S1/S2 cleavage efficiency with S protein fusogenicity
The SARS-CoV-2 S protein S1/S2 cleavage is pivotal, affecting viral fusogenicity 28 .For example, the level of Delta S protein S1/S2 cleavage is higher than that of the B.1.1 and BA.1 S proteins, being associated with higher fusogenic potential in the Delta variant 21,23,25,29 .
To investigate whether the relationship between S protein S1/S2 cleavage and its fusogenicity could apply to S proteins from the other SARS-CoV-2 variants, we subjected HEK293 cells expressing each S protein to western blotting and quantified the full-length S and cleaved S2 band intensities (Fig. 2A, 2B).Full-length S and S2 band intensity variations indicated that each S protein exhibits a different susceptibility to cellular protease-induced S1/S2 cleavage.Consistent with previous results 21,23,25 , the Delta and Lambda S proteins displayed the highest S1/S2 cleavage efficiencies (Fig. 2A, 2B).In addition, the Wuhan, BA.1, BA.2, and BA.5 S protein S1/S2 cleavage was significantly lower than that of the B.1.1 S protein (Fig. 2A, 2B).Finally, we investigated how fusogenicity and S1/S2 cleavage efficiency correlate among the eleven SARS-CoV-2 variants (Fig. 2C).Remarkably, the correlation coefficient (R 2 = 0.5975) indicated a strong positive correlation between fusogenicity and S1/S2 cleavage efficiency of all eleven SARS-CoV-2 S protein variants (Fig. 2C).In summary, these results demonstrated that SARS-CoV-2 S protein fusogenicity is strongly associated with S protein S1 and S2 subunit cleavage.

Correlation of infection-induced plaque size with S protein fusogenicity
The Delta variant with higher fusogenic S protein levels reportedly forms larger plaques than the B.1.1 variant in infected VeroE6/TMPRSS2 cells 21 .However, the BA.1 variant encoding the lower fusogenic S gene displays smaller plaques compared to the B.1.1 variant 23 .These results suggest that S protein fusogenicity is potentially associated with viral infection, indicated the size of the plaques formed by clinical isolates.To address this question, we performed plaque assays using the eleven clinical isolates including the previous VOCs and VOIs and compared the plaque diameters formed by each clinical isolate in VeroE6/TMPRSS2 cells (Fig. 3A, 3B).We observed that the Wuhan, Alpha, Beta, Gamma, Delta, Lambda, and Mu variants formed significantly larger plaques than B.1.1 (Fig. 3A, 3B).

Correlation of SARS-CoV-2 S pseudovirus infectivity with S protein fusogenicity
Next, we examined the correlation of S protein-mediated cell-free viral infection with S protein fusogenicity.We used HEK293T cells to produce HIV-1 virions pseudotyped with SARS-CoV-2 S protein carrying the C-terminal deletion of 19 amino acids (∆19CT), normalized by the p24 concentration, and measured viral infectivity as described previously [20][21][22][23][24]26,27,[29][30][31] . Our resultsrevealed that the Delta and BA.5 S pseudoviral infectivity measured using the HOS-ACE2/TMPRSS2 system were significantly higher than that of the B.1.1 S pseudovirus while that of the Wuhan, Alpha, Beta, Lambda, Mu, BA.1 and BA.2 S pseudoviruses significantly decreased in HOS-ACE2/TMPRSS2 cells compared to B.1.1 (Fig. 4A).We observed no statistically significant difference between the Gamma S pseudoviral infectivity and that of the B.1.1 S pseudovirus (Fig. 4A).Notably, we noticed that the infectivity degrees of the eleven SARS-CoV-2 variant-derived S protein-mediated cell-free pseudoviruses did not correlate with the fusogenicity mediated by these S proteins (Fig. 4B, R 2 = 0.0033).This discrepancy suggests that the each S protein incorporation level into the pseudoviruses might vary, thereby leading to incorrect viral infectivity validation.To test this hypothesis, we analyzed the S protein incorporation levels in the viral particles using western blotting (Fig. 4C, 4D).The S protein band intensity quantification revealed that compared to the B.1.1 S pseudovirus, Wuhan, Alpha, Mu, BA.1, BA.2, and BA.5 variantderived S proteins incorporated less in the pseudovirus particles (Fig. 4C, 4D).These data indicate variable S protein incorporation levels, prompting that viral infectivity per incorporated S protein should be evaluated.Although most SARS-CoV-2 S pseudoviral infectivity divided by S protein incorporation levels became comparable or lower than that of the B.1.1 S pseudovirus, only those of the BA.2 and BA.5 S pseudoviruses were significantly higher (Fig. 4E).In addition, the R 2 value between the cell-free pseudoviral infectivity per S protein and fusogenicity mediated by these S proteins slightly changed from 0.0033 to 0.1482 (Fig. 4B, 4F).In summary, these data suggest that S protein fusogenicity might have little effect on pseudovirus infection in target cells.

Correlation of SARS-CoV-2 S pseudovirus entry efficiency with S protein fusogenicity
The aforementioned pseudovirus assays indicated a weak correlation between S protein fusogenicity and SARS-CoV-2 S pseudoviral infectivity even after normalizing the S protein incorporation levels into the viral particles (Fig. 4F).These data suggest that HIV-1 infection mechanisms, such as reverse transcription and integration, might affect the pseudovirus assay results.To address this hypothesis, we performed a BlaM-Vpr assay using S protein with ∆19CT enabling us to quantify cell-free virus entry efficiency into HOS-ACE2/TMPRSS2 cells (Fig. 5A).The results indicated that all S pseudoviruses exhibited comparable or lower entry efficiencies compared to the B.1.1 S pseudovirus (Fig. 5A).Similar to the pseudovirus assays (Fig. 4B, 4C), we normalized the fluorescence signals indicating entry efficiency into the target cells by the S protein incorporation levels due to the variable S protein incorporation levels into the viral particles (Fig. 5B-D).Interestingly, although the entry efficiency determined by BlaM-Vpr assays and normalized by the viral S protein level strongly correlated with the S pseudovirus infectivities per S protein incorporation levels (Fig. 5E; R 2 = 0.6209), the relationship with S protein fusogenicity was weak, consistently with pseudoviral infectivity (Figs.4F, 5F; R 2 = 0.1482 and 0.0014, respectively).These data support the results suggesting that the S protein fusogenic potential could affect little pseudovirus entry into the HOS-ACE2/TMPRSS2 cells.

Correlation of replication kinetics of clinical isolates in VeroE6/TMPRSS2 and Calu-3 cells with S protein fusogenicity
To investigate the clinical isolate replication kinetics, we inoculated the eleven SARS-CoV-2 variants into VeroE6/TMPRSS2 and Calu-3 cells and measured the viral RNA levels in the cell culture supernatants using RT-qPCR (Fig. 6A, 6B).Compared to the B.1.1 variant, the Lambda, BA.1, and BA.5 variants displayed slower replication kinetics in VeroE6/TMPRSS2 cells while the other variants were comparable (Fig. 6A).In Calu-3 cells, the Alpha, Gamma, Delta, Mu, and BA.5 variant replication kinetics indicated significantly higher and that of the BA.2 variant lower values than that of the B.1.1 variant (Fig. 6B).The other variants, such as Wuhan, Beta, Lambda, and BA.1, displayed no statistically significant difference (Fig. 6B).Finally, we aimed at addressing a potential correlation between viral RNA production in VeroE6/TMPRSS2 or Calu-3 cells 48 hours postinfection and S protein fusogenicity (Fig. 6C, 6D).However, we obtained low R 2 values, indicating no and weak viral RNA production correlation with S protein fusogenicity (Fig. 6C, 6D; R 2 = 0.0152 in VeroE6/TMPRSS2 cells and R 2 = 0.1543 in Calu-3 cells, respectively).These data suggest that S protein fusogenicity might not or could only slightly affect viral RNA production during viral replication in VeroE6/TMPRSS2 and Calu-3 cells.

Discussion
SARS-CoV-2 constantly evolves with mutations in its viral genome since its emergence in late 2019.In particular, the virological characteristics of the Omicron variants, such as transmissibility, pathogenicity, and immunity resistance, are rather different from those of the pre-Omicron variants 11,15,18,19,32,33 .The S gene is the most variable gene in the emerged SARS-CoV-2 variants.The SARS-CoV-2 S protein is pivotal in mediating the virus-target cell membrane fusion.Previous studies indicated that the SARS-CoV-2 S protein fusogenicity is closely associated with intrinsic pathogenicity [20][21][22][23][24] .Therefore, describing S protein features is of utmost importance.In this study, we investigated the correlation between S protein fusogenicity and other virological parameters in eleven SARS-CoV-2 variants including previous SARS-CoV-2 VOCs and VOIs.The S protein fusogenicity strongly correlated with S protein S1/S2 cleavage in transfected HEK293 cells (Fig. 2C) and the plaque size in VeroE6/TMPRSS2 cells infected by clinical isolates (Fig. 3C).However, the S protein fusogenicity was weakly associated with S protein-mediated pseudoviral infectivity (Figs.4B, F) and entry efficiency in HOS-ACE2/TMPRSS2 cells (Fig. 5F) as well as viral replication kinetics in VeroE6/TMPRSS2 and Calu-3 cells (Figs. 6C, D).Taken together, our data suggest that, similar to SARS-CoV-2 S protein fusogenicity, S1/S2 cleavage and plaque size could be potential indicators to predict the intrinsic pathogenicity of newly emerged SARS-CoV-2 variants.
Newly emerged SARS-CoV-2 variant pathogenicity is rather different in clinical settings before and after the emergence of the Omicron BA.1 variant 18 .Although the Delta variant resulted in more severe outcome in infected patients, those of the Omicron BA.1 variant were attenuated 18 .Newly emerged SARS-CoV-2 variant pathogenicity might be predictable by measuring S protein fusogenicity as the latter is reportedly closely associated with the intrinsic pathogenicity of certain SARS-CoV-2 variants [20][21][22][23][24] .For example, the Delta variant displays more fusogenic S proteins and represents higher pathogenicity as demonstrated in a hamster model 21 .In contrast, the S protein of the Omicron BA.1 variant is less fusogenic and its pathogenicity also remains relatively low 23,24 .In support of these clinical and experimental observations, our data demonstrated that the S proteins of six pre-Omicron variants (Alpha, Beta, Gamma, Delta, Lambda, and Mu) exhibited higher fusogenicity than the D614G S protein based on our S protein-mediated membrane fusion assay, while the Omicron S proteins of the BA.1, BA.2 and BA.5 variants yielded comparable or lower fusogenicities (Fig. 1B).Moreover, we demonstrated that the S protein S1/S2 cleavage efficiency and plaque size in clinical isolates correlated with S protein fusogenicity (Figs.2C, 3C), suggesting that these parameters could serve as potential markers for intrinsic pathogenicity prediction of newly emerged SARS-CoV-2 variants.
Nevertheless, the relationship between viral fusogenicity and viral intrinsic pathogenicity is not applied to recently emerged Omicron subvariants, the BQ.1.1 and XBB.1 variants 26,27 .Although these variants demonstrated higher fusogenicity than the BA.5 and BA.2.75 variants, their intrinsic pathogenicity was comparable or lower 26,27 .These unexpected results might be explained by a scenario where non-S proteins could cancel the S protein-induced viral intrinsic pathogenicity increase.In fact, ORF1ab [34][35][36][37] , ORF3a [38][39][40] , ORF6 38,41 , ORF7a 42 , and ORF8 [43][44][45] gene deletions or mutations and those of genes downstream of the S gene 46 are reportedly associated with viral growth attenuation in cell lines or pathogenicity in infected animal models.Remarkably, the BQ.1.1 and XBB.1 variants display at least six and seven amino acid substitutions in the non-S protein coding region compared to the BA.5 and BA.2.75 variants 26,27 .One or some of these substitutions might be involved in attenuating the S protein-augmented intrinsic pathogenicity.Further studies would be required to investigate the non-S gene-intrinsic pathogenesis interaction.SARS-CoV-2 S protein fusogenicity is essential for viral entry into the target cells.
Nevertheless, SARS-CoV-2 S pseudoviral infectivity, entry efficiency, and viral replication indicated less correlation with S protein fusogenicity (Figs.4F, 5F, 6C, 6D).Concerning our pseudovirus and BlaM-Vpr assays, we used lentiviruses pseudotyped with different S proteins of interest.In addition, we used HOS-ACE2/TMPRSS2 cells as non-natural SARS-CoV-2 target cells that stably express ACE2 and TMPRSS2.The related observations could contribute to drawing a difference from viral infection in vivo.Furthermore, viral RNA production during viral replication in VeroE6/TMPRSS2 and Calu-3 cells did not correlate with S protein fusogenicity.Indeed, various studies described that at least pseudoviral infectivity is not necessarily consistent with viral fusogenicity, pathogenicity, and epidemiology 22,24,26,27,44,[47][48][49][50] .Accordingly, our pseudoviral infectivity, BlaM-Vpr, and viral replication assay-related data might be reasonable.However, performing these assays in at least primary lung cells would need to better understand how virological parameters (e.g., SARS-CoV-2 S pseudoviral infectivity, entry efficiency, and viral replication kinetics) could affect viral pathogenicity.
In summary, we revealed that S protein S1/S2 cleavage efficiency and clinical isolate plaque size are associated with SARS-CoV-2 S protein fusogenicity.Since the S protein fusogenicity is closely associated with intrinsic viral pathogenicity, these virological parameters could be used as potential markers to predict the intrinsic pathogenicity of newly emerged SARS-CoV-2 variants.However, the relationship between viral fusogenicity and intrinsic pathogenicity do not apply to the BQ.1.1 and XBB.1 variants.Therefore, further studies would be required to precisely describe the virological features of newly emerged SARS-CoV-2 variants, such as viral pathogenicity-determining factor(s).
Virus was diluted in virus dilution buffer [1M HEPES, DMEM (low glucose), Non-essential Amino acid (gibco, Cat# 11140-050), 1% P/S] and the dilution buffer containing virus was added to the flask after removing the initial medium.After 1 hour of incubation at 37°C, the supernatant was replaced with 15 ml of 2% FBS/DMEM (low glucose) and cell culture was continued to incubate at 37°C until visible cytopathic effect (CPE) was clearly observed.
Then, cell culture supernatant was collected, centrifuged at 300 × g for 10 minutes and frozen at -80°C as working virus stock.The titer of the prepared working virus was determined as the 50% tissue culture infectious dose (TCID50) 20,26,27 .The day before infection, VeroE6/TMPRSS2 cells (10,000 cells) were seeded in a 96-well plate and infected with serially diluted working virus stocks.The infected cells were incubated at 37°C for 4 days and the appearance of CPEs in the infected cells was observed by a microscope.The value of TCID50/ml was calculated by the Reed-Muench method 57 .
The expression level of the surface S proteins was detected by FACS Canto II (BD Biosciences) and analyzed by FlowJo software v10.7.1 (BD Biosciences) (Fig. 1A).RL activity was normalized to the mean fluorescence intensity (MFI) of surface S proteins, and the normalized values are shown as fusion activity (Fig. 1B).
After 1 hour, the cell culture supernatant including pseudoviruses was replaced with warmed fresh DMEM (high glucose), followed by incubation for 3 hours at 37°C.Then, each well was washed with phosphate-buffered saline (PBS) and β-lactamase loading solution (Invitrogen,

Figure legends
Cat# K1030) [CCF4 substrate, solution B, solution C and anion transport inhibitor (Invitrogen, Cat# K1156) in Opti-MEM] was loaded.The plate was covered with aluminum foil to avoid light exposure and kept for 2 hours at room temperature.After 2 hours, cells were harvested, washed with PBS twice, and fixed with 2% paraformaldehyde phosphate (Nacalai Tesque, Cat# 09154-85).The fluorescence intensities at 520 nm (Pacific Blue; uncleaved CCF4) and 447 nm (AmCyan; cleaved CCF4) were detected by FACS Canto II (BD Biosciences) and analyzed by FlowJo software v10.7.1 (BD Biosciences).The ratio of cleaved CCF4 signal to the sum of uncleaved and cleaved CCF4 signals was calculated and shown as entry efficiency (Fig. 5A, 5D).

Fig. 1 .
Fig. 1. S protein fusogenicity correlates with S1/S2 cleavage efficiency (A) Surface S protein expression in transfected HEK293 cells.The data represent the mean fluorescence intensity (MFI) with the mean ± standard deviation (SD) from 3 independent experiments.The statistical significance was tested against B.1.1 (black bar) using twosided unpaired t-test.The P-values are indicated above each bar.The gray and blue bars indicate no significance and significantly reduced expression levels, respectively.(B) Indicated S variant fusion activities.The S protein-mediated membrane fusion assay was performed in Calu-3/DSP1-7 cells.The data at each time point are represented as the mean ± SD from 4 independent experiments.The numbers in each graph indicate the fold change 24 hours after S protein-expressing HEK293 and Calu-3 cell coculture compared to those expressing the B.1.1 S protein (black line).Statistical differences between B.1.1 S and each S variant across timepoints determined by multiple regression.The P-values are indicated

Fig. 2 .Fig. 3 .
Fig. 2. S protein fusogenicity correlates with S1/S2 cleavage efficiency (A) Western blot analysis of S protein expressed in transfected HEK293 cells.The S and S2 proteins were detected using a rabbit anti-SARS-CoV-2 S2 polyclonal antibody.We used anti-α-tubulin as a loading control.(B) Indicated S variant S/S2 cleavage efficiencies.Each bar represents the S/(S + S2) values with the mean ± standard deviation from 5 independent experiments.The statistical significance was tested against B.1.1 (black bar) using twosided unpaired t-tests.The P-values above each bar indicated in blue (reduction), red (increase), or gray (not significant).(C) Correlation between the S variant S/S2 cleavage

Fig. 4 .
Fig. 4. SARS-CoV-2 S pseudovirus infectivity does not correlate with S protein fusogenicity (A) Representative SARS-CoV-2 pseudoviral infectivity data.We used HEK293T cells to produce the HIV-1-based pseudoviruses with the indicated SARS-CoV-2 S proteins for HOS-ACE2/TMPRSS2 cell infection.We measured the intracellular luciferase activity of each virus-infected cell and expressed as viral infectivity relative to that of B.1.1 S pseudovirus (black bar) with the mean ± standard deviation (SD) from three independent experiments.The statistical significance was tested against B.1.1 S using two-sided unpaired t-tests.The P-values above each bar are indicated in blue (reduction), red (increase), or gray (not significant).(B) Correlation between relative pseudoviral infectivity and fusion activity 24 hours after coculture.(C) Representative western blot data of S proteins incorporated in the pseudoviruses.S and S2 were detected using a rabbit anti-SARS-CoV-2 S2 polyclonal antibody.We used p24 as a loading control.(D) S protein incorporation levels in the pseudoviruses relative to B.1.1.The data are represented as the mean ± SD from 4 independent experiments.The statistical significance was tested against

Fig. 5 .
Fig. 5. S protein-mediated pseudovirus entry efficiency does not correlate with S protein fusogenicity (A) Representative BlaM-Vpr assay data.We used HEK293T cells to produce HIV-1-based pseudoviruses with the indicated SARS-CoV-2 S proteins for HOS-ACE2/TMPRSS2 cell infection.The cleaved CCF4 signal ratio to the sum of uncleaved and cleaved CCF4 signals was calculated and indicated as the entry efficiency relative to that of the B.1.1 S pseudovirus (black bar) with the mean ± standard deviation (SD) from three independent experiments.The statistical significance was tested against B.1.1 S using two-sided unpaired t-tests.The P-values above each bar are indicated in blue (reduction) or gray (not significant).(B) Representative western blot data of S proteins incorporated in the pseudoviruses.S and S2 were detected using a rabbit anti-SARS-CoV-2 S2 polyclonal antibody.We used p24 as a loading control.(C) S protein incorporation level in the pseudoviruses relative to B.1.1.The data are represented as the mean ± SD from 4 independent experiments.The statistical significance was tested against B.1.1 S (black bar) using two-sided paired t-tests.The P-values above each bar are indicated in blue (reduction)